Ionization source for mass spectrometry analysis

ABSTRACT

A new ionization source named Surface Activated Chemical Ionization (SACI) has been discovered and used to improve the sensitivity of the mass spectrometer. According to this invention the ionization chamber of a mass spectrometer is heated and contains a physical new surface to improve the ionization process. The analyte neutral molecules that are present in gas phase are ionized on this surface. The surface can be made of various materials and may also chemically modified so to bind different molecules. This new ionization source is able to generate ions with high molecular weight and low charge, an essential new key feature of the invention so to improve sensitivity and reduce noise. The new device can be especially used for the analysis of proteins, peptides and other macromolecules. The new invention overcomes some of the well known and critical limitations of the Electrospray (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometric techniques.

FIELD OF THE INVENTION

This invention relates to the field of mass spectrometry, and moreparticularly to improvements in the chemical ionization source to beapplied to mass spectrometers.

BACKGROUND OF THE INVENTION

A variety of ionization sources, for the analysis of molecules withmedium-high molecular weight (like peptides and proteins) are essentialcomponents of modern mass spectrometric instruments. The ionizationsource transforms neutral molecules into ions which can be analyzed bymass spectrometry.

A mass spectrometer generally has the following components:

(1) a device, usually a Liquid Chromatograph, for the separation orde-salting of the molecules contained in a sample;

(2) an ionization source, contained in a chamber, to produce ions fromthe analyte;

(3) at least one analyzer or filter which separates the ions accordingto their mass-to-charge ratio;

(4) a detector that counts the number of the ions;

(5) a data processing system that calculates and plots a mass spectrumof the analyte.

The mass spectrometry techniques currently used for the analysis ofmacromolecules and, especially, proteins and peptides are based on theElectrospray Ionization (ESI) (U.S. Pat. No. 5,756,994; Cunsolo V, FotiS, La Rosa C, Saletti R, Canters G W, Verbeet M. Ph. Rapid Commun. MassSpectrom. 2001; 15: 1817; Wall D B, Kachman M T, Gong S S, Parus S J,Long M W, Lubman D M. Rapid Commun. Mass Spectrom. 2001; 15: 1649;Fierens C, Stöckl D, Thienpont L M, De Leenheer A P. Rapid Commun. MassSpectrom. 2001; 15: 1433; Li W, Hendrickson C L, Emmett M R, Marshall AG. Anal. Chem. 1999; 71: 4397; Fierens C, Stöckl D, Thienpont L M, DeLeenheer A P. Rapid Commun. Mass Spectrom. 2001; 15: 451) and MatrixAssisted Laser Desorption Ionization (MALDI) (U.S. Pat. No. 5,965,884;Cozzolino R, Giorni S, Fisichella S, Garozzo D, La fiandra D, Palermo A.Rapid Commun. Mass Spectrom. 2001; 15: 1129; Madonna A J, Basile F,Furlong Ed, Voorhees K J. Rapid Commun. Mass Spectrom. 2001; 15: 1068;Basile A, Ferranti P, Pocsfalvi G, Mamone G, Miraglia N, Caira S,Ambrosi L, Soleo L, Cannolo N, Malorni A. Rapid Commun. Mass Spectrom.2001; 15: 527; Galvani M, Hamdan M, Rigetti P G. Rapid Commun. MassSpectrom. 2001; 15: 258; Ogorzalek Loo R R, Cavalcali J D, VanBogelen RA, Mitchell C, Loo J A, Moldover B, Andrews P C. Anal. Chem. 2001; 73:4063).

Both techniques are highly effective for the production of ions ofbiomolecules in the gas phase, to be subsequently analyzed by MassSpectrometry (MS).

In the case of ESI, multicharge ions of medium/high molecular weightcompounds are produced. The mass of macromolecule compounds is thenobtained using specific software algorithms.

Mass spectrometry represents an essential technology in the analyticalfield. It is usually coupled with other separative techniques, so as toidentify chemical compounds and quantify complex biological mixtures.Proteins, for instance, are first separated, collected and then digestedwith Trypsin. The masses of the resulting peptides are determined bymass spectrometry (normal scan MS or tandem mass spectrometry MS/MS). Inthe case of the MS/MS approach, peptide ions of a single m/z ratio arefragmented by collision induced dissociation (CID) and then analyzedusing various mass analyzers (triple quadrupole, ion trap, Fouriertransform-ion cyclotron resonance). Each peptide gives origin tospecific mass patterns for a given amino acid sequence. The peptidesequences can be obtained by computer analysis of the data using adedicated software (database search and de novo sequence software). Inorder to obtain good MS/MS spectra doubly charge peptide ions arepreferably fragmented (Cramer R, Corless S. Rapid Commun. Mass Spectrom.2001; 15: 2058). The electrospray and MALDI techniques when are appliedto the analysis of peptides with high molecular weight (2000-4000Thompson (Th)) using the MS/MS approach have some limitations. Forinstance, when proteins or peptides with high molecular weight areanalyzed, ESI multicharge ions are produced. These ions give rise tocomplex fragmentation spectra, difficult to interpret. For this reasononly peptides with a maximum of 15 amino acidic residues can be analyzedby tandem mass spectrometry. In the case of MALDI only mono-charge ionsare usually obtained. If the MALDI source is coupled with Time of FlightMass Analyzer (TOF) the technique used to fragment the ions is the postsource decay (PSD). This fragmentation technique give rise to someadditional problems; in order to obtain good fragmentation spectra it isusually necessary to use peptide derivatization. A MALDI atmosphericpressure source has recently been coupled with an ion trap analyzer.This configuration makes possible the structural analysis of peptides byMS/MS and MS³. However, it must be emphasized that the MALDI sourceproduces, mainly, mono-charge peptide ions that produce fragmentationspectra more complex and less specific than those obtained byfragmentation of the bi-charge ions.

Another problem that affect both MALDI and ESI techniques is representedby the decrease in sensitivity when salts are present in the sample. Inthe case of ESI the problem may be solved by coupling the massspectrometer with a pre-analytical separation step, such as by the useof an High Performance Liquid Chromatographer (HPLC) or other de-saltingtechniques. This obviously introduces another step in the wholeprocedure of analysis. The HPLC technique on the other hand cannot beused for the case of MALDI because in this case it is necessary toco-crystallize the analyte with a matrix molecule. Salts contained inthe sample must, however, be eliminated before of the crystallizationstep by well known additional treatments of the sample.

PURPOSE AND DESCRIPTION OF THIS INVENTION AND IMPROVEMENTS OVER THEPRIOR ART

The present invention is based on the introduction of a device for theionization of neutral molecules in the gas phase. The device comprisesan active surface carrying element that, according to this invention, isinserted in the ionization chamber. This technique has been named by us“Surface Activated Chemical Ionization” (SACI). SACI technique allowsthe ionization to be performed at atmospheric pressure.

Use of an atmospheric-pressure ionization has already been proposed andis known as the APCI technique. APCI instrument makes use of aneedle-shaped corona discharge electrode inserted inside the ionizationchamber. However, the high energy of the corona discharge electrodeleads to the macromolecules fragmentation. The main problem of thismethod is the lower sensitivity with respect to ESI and MALDItechniques.

We have now surprisingly found that introducing into the ionizationchamber a plate-like active-surface carrying element can bring tounexpected results in term of high sensitivity and possibility to detectmolecules having a molecular weight in a broad range of values.

According to the invention, the solution containing the analyte isinjected in the SACI source through an inlet aperture. The sample isnebulized by a gas flow and vaporized by heating. The ionization chambercontains an active surface carrying element onto which the vaporizedmolecules of the analyte bump, so that the analyte becomes ionized. Thisactive surface can be made of various materials (steel, glass, quartzetc), both electrically conductive or not. Different molecules can alsobe bound or absorbed over the surface to improve the ionization process(H₂, D₂O and various acid and basic molecules). The analyte neutralmolecules which are present in gas phase are ionized by variousphysical-chemical interactions which take place on the surface. Surfaceproperties and function in catalyzing various kind of reactions is wellknown (U.S. Pat. No. 5,503,804; U.S. Pat. No. 5,525,308; U.S. Pat. No.5,856,263; U.S. Pat. No. 5,980,843).

An interesting use of a surface in mass spectrometry is the SurfaceEnhanced Laser Desorption Ionization (SELDI) (U.S. Pat. No. 6,020,208;U.S. Pat. No. 6,124,137; U.S. Patent No. 20020060290; U.S. Pat. No.5,719,060). In this case the probe of MALDI mass spectrometer carries animmobilized affinity reagent which binds the analyte on its surface.Furthermore an energy absorbing material is added to the dried sampleand Laser Desorption Ionization mass spectrometry is used to analyze thesample. This technique however differs from the SACI because of the factthat the sample can be prepared in advance by deposition over thesurface, so that this analysis is quite time consuming. Some ionizationsource make use of an electrical potential applied to a needle to ionizethe sample, in gas phase, by using the corona discharge effect (U.S.Pat. No. 6,407,382; U.S. Pat. No. 5,684,300; U.S. Pat. No. 6,294,779;U.S. Pat. No. 5,750,988; U.S. Pat. No. 6,225,623; U.S. Pat. No.5,756,994; U.S. Pat. No. 20020074491; U.S. Pat. No. 20020048818; U.S.Pat. No. 20020011560; U.S. Pat. No. 4,849,628).

The use of the SACI ionization source which is disclosed in thisinvention, represents a key improvement for the production of ions withhigh molecular weight and low charge (bi-charge ions are usually muchabundant). The innovative aspect of this invention over the previousknown art can be so summarized:

a) Analytes with higher molecular mass can be studied since thetechnique is able to generate ions with high molecular weight and lowcharge, an essential feature useful for obtaining the mass ofmacromolecule compounds. Best results can be obtained if the source iscoupled with a mass analyzer with high mass range like FourierTransform—Ion Cyclotron Resonance (FT-ICR) or Time Of Flight (TOF).

b) A higher sensitivity can be obtained in the analysis of moleculeswith high mass and low charge (typically bi-charge ions). This isparticularly useful for analyzing biological compounds, like proteinsand peptides, which are frequently present at low concentration inbiological samples (tissues, urine, etc).

c) The new technique makes it now possible to analyze molecules withmedium/high mass and low charge (typically the bi-charge ions), by theMS/MS approach. This feature is useful to characterize proteins and highmolecular weight peptides. In fact we have shown that peptidescontaining more than 15 amino acidic residues can be studied. This isparticularly useful for the characterization of peptides with high mass,originated by missed cleavage during the enzymatic digestion reaction.

d) The SACI ionization source is much less affected by the presence ofsalts than the ESI and MALDI sources. The new invention makes it nowpossible to analyze liquid biological samples, which usually containsalts or buffers, by direct infusion into the mass spectrometer withoutusing an HPLC systems or other desalting procedures. This isparticularly useful for analyzing samples in high throughputapplications. Samples containing a high concentration of salts are wellknown to give rise to serious problem when the ESI or MALDI techniquesare used.

Table 1 summarize the critical improvements obtained by the applicationof SACI vs ESI technique.

TABLE 1 A summary of the critical improvements obtained by theapplication of SACI vs ESI techniques SACI vs ESI Detect ions with highDetect multicharge ions mass and low charge with high mass Highthroughput Pre-analytical steps limit “Tolerant” of salts throughput Cansequence peptides with Less tolerant of salts high molecular weight Cannot sequence peptides (more than 15 amino acid) longer than 15 aminoacid High sensitivity, Higher chemical noise Low chemical noise Lowersensitivity

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A schematic representation of the new device, i.e. the SurfaceActivated Chemical Ionization source (SACI).

FIG. 2:

a) Mass spectrum, obtained by direct infusion in the mass spectrometerusing the SACI technique, of a sample containing a mixture of fivepeptides (peptide YY fragments 13-36 obtained from Sigma catalog numberP6613, MW 3014 Da; Diabetes associated peptide fragment 8-37 obtainedfrom Sigma catalog number. D6170, MW 3200 Da; Gastrin releasing peptidehuman obtained from Sigma catalog number G8022, MW 2859 Da;Phospholipase 2 activating peptide obtained from Sigma catalog numberG1153, MW 2330 Da; and Vasoactive Intestinal Peptide Fragment 6-28obtained from Sigma catalog number V4508, Mw 2816 Da) acquired in the400-4000 Th range. The solution concentration of each peptide was 10⁻⁷M. The counts/s value was 10⁶ and the S/N ratio of the most abundantpeak was 500. No salts were added in the pure H₂O solution containingthe peptides.

b) Mass spectrum, obtained by direct infusion in the mass spectrometerusing the ESI technique, of the same solution as in (a). The counts/svalue was 10⁵ and the S/N ratio of the most abundant peak was 100. Amuch higher chemical noise can be observed in this case, leading to adecrease of the S/N ratio. Using the SACI ionization source the mono andbi-charge ions were mainly obtained, whereas using the ESI ionizationsource only the tri-charge ions can be detected. It must be emphasizedthat the multicharge phenomenon, which takes place by using the ESIsource, leads to a compression of the mass signals. An overlap of themulticharge signals, which usually takes place for molecules with highmolecular weight is also observed.

FIG. 3:

a) Mass spectrum, obtained by direct infusion in the mass spectrometerusing the SACI technique, of a standard protein (Cytochrome C) acquiredin the 4000-14000 Th range. The protein was obtained by Sigma-Aldrich(catalog number 10,520-1) and diluted in H₂O so to obtain aconcentration of 10⁻⁷ M. The counts/s value was 10⁶ and the S/N ratio ofthe most abundant peak was 300.

b) Mass spectrum obtained by direct infusion in the mass spectrometerusing the ESI technique, of the same solution as in (a). No signals weredetected in this case. This is due to the extensive multichargephenomenon that takes place in the ESI ionization source.

c) Multicharge distribution of the Cytochrome C protein obtained usingthe ESI ionization source. The multicharge distribution is usuallycompressed in the first region of the spectrum (100-2000 Th) thusleading to a decrease of the sensitivity.

FIG. 4:

a) Tandem mass spectrum, obtained by using the SACI technique, of thebi-charge ion of Vasoactive Intestinal Peptide Fragment 6-28 at m/z1409.

b) Tandem mass spectrum of the same solution, obtained using the ESItechnique. The tri-charge ion at m/z 940 was fragmented. In the case ofthe fragmentation of the tri-charge ion few fragmentation peaks wereobtained.

FIG. 5:

a) Mass spectrum, obtained by direct infusion in the mass spectrometerusing the SACI technique, of a sample containing a mixture of fivepeptides, as in FIG. 2 a, acquired in the 400-4000 Th range. Thesolution had a ammonium bicarbonate (NH₄HCO₃) concentration of 50mmol/L. The counts/s value was 10⁶ and the S/N ratio of the mostabundant peak was 500.

b) Mass spectrum obtained by direct infusion in the mass spectrometerusing the ESI technique, of the same solution as in (a). The counts/svalue was 10⁵ and the S/N ratio of the most abundant peak was 100. Inthe case of the ESI technique a high chemical noise leads to decreasethe quality of the spectrum. The multicharge phenomenon also takes placeleading to decrease the quality of the spectrum.

FIG. 6:

a) Mass spectrum, obtained by direct infusion in the mass spectrometerusing the SACI technique, of a peptide mixture obtained by trypticenzymatic digestion of Cytochrome C, in the presence of 50 mmol/LNH₄HCO₃. The identified peptides are marked by their amino acidicintervals as compared with the original protein sequence. The initial(before tryptic digestion) concentration of the protein was 10⁻⁷ M. Thecounts/s value was 10⁶ and the S/N ratio of the most abundant peak was450.

b) Mass spectrum, obtained by direct infusion in the mass spectrometerusing the ESI technique, of the same solution. The counts/s value was10⁵ and the S/N ratio of the most abundant peak was 100. In this case ahigher chemical noise as compared with (a) is present. Moreover, in thecase of the ESI ionization source spectrum, less peptide signals weredetected.

FIG. 7:

a) Mass spectrum, obtained by direct infusion in the mass spectrometerusing the SACI technique and in absence of salts, of a sample containinga mixture of five peptides as in FIG. 2 a. The counts/s value was 10⁶and the S/N ratio of the most abundant peak was 500.

b) Mass spectrum obtained by direct infusion in the mass spectrometerusing the SACI technique, of a sample containing a mixture of fivepeptides as in (a), but containing 50 mmol/L NH₄HCO₃. It must beemphasized that this buffer is commonly used for biological application(for example to perform the tryptic digestion). The counts/s value was10⁶ and the S/N ratio of the most abundant peak was 500. It should benoted that the presence of the buffer does not lead to a decrease in thequality of the spectrum or a higher chemical noise.

FIG. 8:

a) Mass spectrum, obtained by direct infusion in the mass spectrometerusing the ESI technique, of a sample containing a mixture of fivepeptides as in FIG. 2 b. The counts/s value was 10⁵ and the S/N ratio ofthe most abundant peak was 100.

b) Mass spectrum, obtained by direct infusion in the mass spectrometerusing the ESI technique, of the same sample as in (a) but in thepresence of 50 mmol/L NH₄HCO₃. The counts/s value was 10⁵ and the S/Nratio of the most abundant peak was 100. It can be seen that thepresence of the buffer leads a decrease of the peaks at m/z 778, 954,1006 and 1068.

DESCRIPTION OF A PREFERRED EMBODIMENT OF THE PRESENT INVENTION ANDAPPLICATION EXAMPLES

The SACI source described in this invention and schematicallyrepresented in FIG. 1 produces ions that can be analyzed in a massspectrometer. The spectrometer comprises the ionization source, theanalyzer or filter for separating the ions by their mass-to-chargeratio, a detector for counting the ions and a data processing system.Since the structure of the spectrometer is conventional, it will not bedescribed in more detail, but the ionization source device which is thesubject of the present invention. The ionization source of theinvention, on its turn, does not substantially differ, in its structure,from the known devices of this kind, so that a schematic representationthereof will be sufficient for the skilled man in this art to understandhow it is constructed and works.

The ionization source device of the invention comprises an inletassembly 11 which is in fluid communication with an ionization chamber3.

The ionization chamber 3 comprises an outlet orifice, generally lessthan 1 mm in diameter, for communicating between the ionization chamberand the analyzer or filter. Generally, the angle between the axis of theinlet assembly 11 and the axis passing through said orifice is about90°, but different relative positions can also be envisaged. Inside theionization chamber 3 is positioned a plate 4. The plate 4 has at leastone active surface 4′ which faces the internal aperture of the inletassembly 11. Preferably, the plate 4 is inclined of an angle whichallows the analyte to be reflected, once ionized, towards the outletorifice bringing to the analyzer or filter, so that the highest numberof ions can reach the analyzer (mirror effect). This will stronglyimprove the sensitivity of the method. The said inclination angle willdepend of course on the relative position of the axes of both inletassembly 11 and outlet orifice. For example, if such axes form an angleof 90°, the element 4 will be 45° inclined.

The plate 4 can have different geometries and shapes, such as squared,rectangular, hexagonal shape and so on, without departing for this fromthe scope of the present invention. It has been found that thesensitivity of the analysis increases when the active surface 4′ isincreased. For this reason, the plate 4 surface will range preferablybetween 1 and 4 cm² and will be generally dictated, as the highestthreshold, by the actual dimensions of the ionization chamber 3. Whilemaintaining the dimension of the plate 4 fixed, the active surface 4′area can be increased in various ways, for example by creatingcorrugations on the surface 4′. In particular cases, such as the casewherein low molecular weight molecules must be analyzed, high electricalfield amplitude is required. In such cases, it may be advantageous toprovide the active surface 4′ with a plurality of point-shapedcorrugations, in order to increase in such points the electrical fieldamplitude.

The plate 4 gas generally a thickness of between 0.05 and 1 mm,preferably of between 0.1 and 0.5 mm.

The active surface 4′ can be made of various materials, either ofelectrically conductive or non-conductive nature. Preferred materialscan be a metal such as iron, steel, copper, gold or platinum, a silicaor silicate material such as glass or quartz, a polymeric material suchas PTFE (Teflon), and so on. When the active surface 4′ is comprised ofa non-conductive material, the body of the plate 4 will be made of anelectrically conductive material such as a metal, while at least a facethereof will be coated with the non-conductive material in form of alayer or film to create the active surface 4′. For example, a stainlesssteel plate 4 can be coated with a film of PTFE. It is in fact importantthat, even if of non-conductive nature, the active surface 4′ besubjected to a charge polarization. This will be achieved by applying anelectric potential difference to the body plate, thus causing apolarization to be created by induction on the active surface 41 too. Onthe other: hand, if the surface 4′ is of electrical conductive nature,the plate 4 does not need to be coated. In this case, a good performanceof the ionization source of the invention can be achieved even withoutapplying a potential difference, i.e. by maintaining the surface 4′ atground potential and allowing it to float.

The plate 4 is linked, through connecting means 5, to a handling means 6that allows the movement of the plate 4 in all directions. The handlingmeans 6 can be moved into the ionization chamber and also can berotated. The connecting means 5 can be made of different electricallyconductive materials and can take various geometries, shapes anddimensions. Preferably, it will be shaped and sized so as to facilitatethe orientation of the plate 4 in an inclined position. In this case,the connecting means 5 will have a step-like shape (as shown in FIG. 1).The plate 4 is electrically connected to power supply means 20 in orderto apply a potential difference to the active surface 4′.

Coming now to the description of the inlet assembly 11, the liquidsample containing the analyte is introduced into the chamber through thesample inlet hole 10. The inlet assembly 11 comprises an internal duct,open outwardly via the said inlet hole 10, which brings to anebulization region 12. The said nebulization region is in fluidcommunication with at least one, typically two gas lines 14, 15(typically, the gas is nitrogen) which intercepts the main flow of thesample with different angles, so that to perform the functions of bothnebulizing the analyte solution (angle >45°) and carrying it towards theionization chamber 3 (angle <45°). Downstream to the said nebulizationregion. 12, a heating region 13 is provided. The heating region 13comprises heating means, such as a heating element connected to a powersupply connector 16. The vaporized analyte is thus heated attemperatures ranging from 200° C. and 450° C., preferably of between250° C. and 350° C. The internal duct of the inlet assembly 11 ends intothe ionization chamber 3 in a position which allows the vaporized andheated analyte to impact the active surface 4′ of the plate 4, where theionization of the neutral molecules of the analyte takes place. Withoutbeing bound to any particular theory, it is likely that a number ofchemical reactions take place on the surface: proton transfer reactions,reaction with thermal electron, reaction with reactive molecules locatedon the surface, gas phase ion molecule reactions, molecules excitationby electrostatic induction. It is also possible that the dipolar solventis attracted from the active surface 4′ by means of the chargepolarization induced on it and so provide a source of protons that reactwith the analyte molecules to form ions. As said before, the plate 4 canbe allowed to float—only if the active surface 4′ is electricallyconductive, since in this case an electron exchange flow can beestablished between the solvent and the surface 4′—or a potentialdifference can be applied. Such a potential difference, as absolutevalue, will preferably be in the range of from 0 and 1000 V (inpractice, can range between −1000 V and +1000 V, depending on the kindof polarization that is required on the active surface 4′), preferablyof from 0 and 500 V, more preferably of from 0 and 200 V. High voltage,such as about 200 V, allows the ionization yield to be increased. Thepossibility given by the present invention device to work both with andwithout a voltage to be applied to the analyte is of pivotal importance.In fact, in some instances, there are molecules that do not suffer astrong electrical field, such as the macromolecules or even some smallmolecules like amphetamines, which degrade in such strong conditions. Ingeneral, it can be said that the absence of a voltage applied to theplate 4 avoids redox reactions to the analyte.

For the reasons seen above, it is important that the solvent in whichthe analyte is dissolved be a dipolar solvent having acidic protons.Preferred solvents are H₂O, alcohols such methanol or ethanol,acetonitrile.

The impact angle of the analyte onto the active surface 4′ will bepreferably 45° or less. Low impact angle values allow a better contactbetween the analyte and the active surface, thus improving theionization performance.

In a preferred embodiment of the invention, the analyte solution alsocontains aminoacids such as glycine, lysine, istidine, aspartic acid andglutammic acid, which have the function of proton donors to promote theanalyte ionization.

The ions so formed are reflected and directed to the analyzer 1 throughthe outlet orifice, as described above.

The essential feature of the invention consists in the introduction of an active surface 4′ in the vaporization chamber 3, that enhances theionization of the neutral analyte molecules present in gas phase. TheSACI can be considered a soft ionization source, which can be ofparticular interest in several applications, such as in the field ofdrugs and anti-doping analysis.

It should be understood that the above description is intended toillustrate the principles of this invention and is not intended to limitany further modifications, which can be made following the disclosure ofthis patent application by people expert in the art.

The following, not limiting, examples are described to illustrate thenovelty and usefulness of the invention.

EXAMPLE 1 The Observation of Ions in the High Mass Range

A 10⁻⁷ M solution of Cytochrome C protein (MW: 12361) has been analyzedby direct infusion. FIG. 3 a shows the protein signals obtained usingthe new SACI ionization source. The mono-charge, bi-charge andtri-charge ions were clearly detected using positive acquisition mode.This compares with results on the same solution achieved by the use ofthe ESI ionization source (FIG. 3 b). In this latter case no multichargedistribution was detected in the 4000-14000 Th range. In fact signalsobtained in this region of the spectrum by the use of the ESI ionizationsource are due to the chemical noise of the solvent. It is well knownthat the ESI ionization source cannot be used to analyze molecules withhigh molecular weight and low charge. Thus the ESI technique has seriouslimits for analyzing biological molecules with high molecular weight(like proteins). In order to overcome this limitation the MALDIionization source is used since. The ionization source of MALDI is ableto produce low charge ions in the range 1000-300000 Th. The applicationof MALDI technique, however, requires co-crystallization of the analytewith a matrix molecule. To ionize the sample a laser light that ismainly adsorbed by the matrix molecule is ordinary used. A microexplosion process (ablation) take place on the surface of the crystaland the excited matrix molecules ionize the sample molecules in gasphase (soft ionization reaction). For this reason a HPLC or similar online separation methods cannot be used in the MALDI approach. It must beemphasized that the SACI ionization source is able, like the MALDIsource, to generate ions with high molecular weight and low charge, but,in addition, it can be coupled in line with HPLC or other separatorymethods.

EXAMPLE 2 An Application of SACI Technique to the Analysis of HighMolecular Weight Peptides

Five high molecular weight standard peptides with molecular mass in the2000-4000 Da range were analyzed. The results obtained using the SACIsource are shown in FIG. 2 a. As can be seen the mono and bi-chargepeptide ions were clearly detected. The peptides were analyzed also by amass spectrometer using the ESI ionization source (FIG. 2 b). In thiscase the tri-charge peptide ions are the most abundant species. Thesespecies are located in a region of the spectrum (500-1100 Th) in whichthe chemical noise is high leading to decrease the S/N ratio.

The mass analyzer used to perform both experiments was an ion trap(LCQ^(XP), ThermoFinnigan, USA) able to detect the signals in the100-4000 Th and 1000-20000 Th range. The mass acquisition range can alsobe extended by coupling the SACI ion source with other kind of massanalyzer (for example TOF or FT-ICR) provided with a high massacquisition range.

EXAMPLE 3 Increase in Sensitivity Provided by the New Ionization Source

The SACI ionization source first described in the present invention ischaracterized by a higher sensitivity, as compared to the ESI technique,in the analysis of liquid samples of proteins and peptides. FIGS. 2 aand 3 a show the spectra obtained by direct infusion of solutions offive high molecular weight peptides (FIG. 2 a) and Cytochrome C (FIG. 3a). A LCQ^(XP) (ThermoFinnigan, USA) provided with SACI ionizationsource was used. The solution concentration of each standard peptide andof the Cytochrome C was 10⁻⁷ M and the counts/s value was 10⁶ with a S/Nratio of the most abundant peak of 500 for the high molecular weightpeptides and 300 for the Cytochrome C protein. The comparison of theseresults with those obtained, for the same solutions, using the ESIionization source (FIGS. 2 b and 3 b) shows that the SACI ionizationsource increases the sensitivity. As can be seen for the case of the ESIspectra of the same high molecular weight peptides (FIG. 2 b) the mostabundant signals (tri-charge ions) are detected in the 500-1100 Thrange, due to the multicharge phenomenon. Furthermore, the chemicalnoise is higher (S/N ratio of the most abundant peak=100) using the ESItechnique than that obtained by the SACI ionization source (S/N ratio ofthe most abundant peak=500).

In the spectrum of the Cytochrome C, obtained by the ESI ionizationsource. (FIG. 3 b), no protein signal has been detected in the4000-14000 Th range. This is due to the extensive multicharge phenomenonthat takes place in the ESI ionization source. For this reason themulticharge distribution is usually compressed in the 100-2000 Th range(FIG. 3 c) where the chemical noise is higher.

EXAMPLE 4 Characterization of High Molecular Weight Peptides

The tandem mass spectrometry (MS/MS) of bi-charge ions, that areabundantly produced by the SACI source, can be further characterized. InFIG. 4 a the SACI-MS/MS spectrum of the bi-charge ion of VasoactiveIntestinal Peptide Fragment 6-28 is shown. The bi-charge ion wasisolated into the ion trap analyzer and fragmented by Collision InducedDissociation (CID). The results of the peptide identification and itsrelative statistical correlation score, by the use of the SEQUESTdatabase search program, were as follows:

Peptide Xcorr DeltCn Vasoactive Intestinal Peptide 3.5382 0.204 Fragment6-28

Xcorr is a spectra correlation score and DeltCn is the 1.0—normalizedcorrelation score. A correctly identified peptide has a value of Xcorrscore higher than 3. The peptide was also analyzed using the ESIionization source (FIG. 4 b). In this case the bi-charge peak at m/z1409 had a too weak intensity to obtain an MS/MS spectrum. Thus, thetri-charge ion at m/z 940 was fragmented. The statistical correlationscore and the DeltCn in this case were as follows:

Peptide Xcorr DeltCn Vasoactive Intestinal Peptide 1.2280 0.608 Fragment6-28

As can be seen by the Xcorr and DeltCn scores so calculated, the peptidecharacterization is statistically more accurate using the SACI-MS/MSspectrum obtained fragmenting the bi-charge ions at m/z 1409.

EXAMPLE 5 Effect of Salts on Sensitivity

FIGS. 5 a and 6 a show the mass spectra of a solution of five standardpeptides and of peptides obtained by Cytochrome C tryptic digestion allin 50 mmol/L NH₄HCO₃ buffer. The SACI ionization source was used. Inboth cases the solution concentration was 10⁻⁷ M. The counts/s value was10⁶ and the S/N ratio was 500 in the case of the high molecular weightpeptides and 450 in the case of Cytochrome C peptides. The resultsobtained using the ESI ionization source is shown in FIGS. 5 b and 6 b.As can be seen in these latter cases the mass spectra show a highchemical noise, due to the presence of the buffer. This leads to adecrease in sensitivity as compared to that obtained by the use of SACIionization source. In fact the counts/s value was an order of magnitudelower (10⁵) and the S/N ratio of the most abundant peak (100) is 5 timeslower.

In order to show that the S/N ratio is not affected by salts, FIG. 7reports the mass spectra of five high molecular weight peptides acquiredwithout (FIG. 7 a) and with (FIG. 7 b) salts in the sample solutions.The SACI ionization source was used in both cases. As can be seen saltsdo not lead to a decrease of the spectrum quality. This fact is veryimportant when biological mixtures are analyzed. In fact these mixturesalmost always contain salts or buffers (as for example NH₄HCO₃ used forthe tryptic digestion) that give rise to well known effect on the ESImass spectra.

FIG. 8 shows the spectra obtained by analyzing the high molecular weightpeptide solutions in absence (FIG. 8 a) and in presence. (FIG. 8 b) ofsalts by the standard ESI technique. In both cases the spectra show ahigher chemical noise than in those obtained using the SACI ionizationsource (respectively shown in FIGS. 7 a and 7 b). The addition of theNH₄HCO₃ buffer to the solution analyzed by the ESI technique decreasethe peptide signals at m/z 1068, 1006, 778 and 954. For this very reasonan HPLC or other separation steps system is coupled with the ESIionization source. A chromatographic analysis, however, takes time andincreases the number of manipulation of the sample before analysis. Thisis a limit especially when many samples must be analyzed.

1. Atmospheric-pressure ionization source device, adapted foratmospheric-pressure ionizing analytes in liquid phase, to be furtheranalyzed by mass spectrometry, comprising (a) an inlet assembly, inwhich the analytes in liquid phase are injected, nebulized and vaporizedby heating; and (b) an atmospheric-pressure ionization chamber withwhich said inlet assembly is in fluid communication, said ionizationchamber being provided with an outlet orifice for communicating betweensaid ionization chamber and an analyzer or filter of a massspectrometer, wherein said atmospheric-pressure ionization chambercomprises a plate having at least one active surface which facesinternal apertures of said inlet assembly and onto which the vaporizedmolecules of the analytes bump and are ionized, said active surfacebeing charge polarized wherein said plate is inclined at an angle whichallows the ionized analyte to be reflected towards the analyzer of themass spectrometer.
 2. The atmospheric-pressure ionization source deviceof claim 1, wherein the said active surface is charge polarized byconnection with power supply means.
 3. The atmospheric-pressureionization source device of claim 1, wherein the said active surface ischarge polarized by induction.
 4. The atmospheric-pressure ionizationsource device according to claim 1, wherein the said plate and the saidat least one active surface are made of an electrically conductivematerial.
 5. The atmospheric-pressure ionization source device accordingto claim 4, wherein the said electrically conductive material is chosenbetween iron, steel, gold, copper or platinum.
 6. Theatmospheric-pressure ionization source device according to claim 4,wherein the said plate is coated with a non-conductive material to formthe said at least one active surface.
 7. The atmospheric-pressureionization source device according to claim 6, wherein the saidnon-conductive material is chosen between a silica or silicatederivative such as glass or quartz or a polymeric material such as PTFE.8. The atmospheric-pressure ionization source device according to claim1, wherein the said at least one active surface is provided withcorrugations.
 9. The atmospheric-pressure ionization source deviceaccording to claim 8, wherein said corrugations are point-shapedcorrugations.
 10. The atmospheric-pressure ionization source deviceaccording to claim 1, therein said angle is 45° when the angle betweenthe axes of both the inlet assembly and the outlet orifice is 90°. 11.The atmospheric-pressure ionization source device according to claim 1,wherein the plate is 0.05 to 1 mm thick, preferably 0.1 to 0.5 mm thick.12. The atmospheric-pressure ionization source device according to claim1, wherein the said plate is linked, through connecting means, to ahandling means that allows the movement of the said plate in alldirections.
 13. The atmospheric-pressure ionization source deviceaccording to claim 12, wherein the said connecting means are made of anelectrically conductive material.
 14. The atmospheric-pressureionization source device according to claim 12, wherein the saidconnecting means are step-like shaped.
 15. The atmospheric-pressureionization source device according to claim 1, wherein the said plate isconnected to power supply means.
 16. The atmospheric-pressure ionizationsource device according to claim 1, wherein the said inlet assemblycomprises an inlet hole for feeding the analyte solution and an internalduct in fluid communication with the said inlet hole, said internal ductcomprising a nebulization region and a heating region and ending intothe said atmospheric-pressure ionization chamber.
 17. Theatmospheric-pressure ionization source device according to claim 16,wherein the said nebulization region is in fluid communication with atleast one gas lines for nebulizing the analyte solution and carrying ittowards the atmospheric-pressure ionization chamber.
 18. Theatmospheric-pressure ionization source device according to claim 17,wherein the said gas is nitrogen.
 19. The atmospheric-pressureionization source device according to claim 1, wherein the said heatingregion comprises heating means, preferably a heating element connectedto a power supply connector.
 20. A mass spectrometer comprising aatmospheric-pressure ionization source device as defined in claim
 1. 21.The mass spectrometer according to claim 20, further comprising: (1) adevice, optionally a Liquid Chromatograph, for the separation orde-salting of the molecules contained in a sample; (2) at least oneanalyzer or filter which separates the ions according to theirmass-to-charge ratio; (3) a detector that counts the number of the ions;(4) a data processing system that calculates and plots a mass spectrumof the analyte.
 22. A method for atmospheric-pressure ionizing ananalyte to be analyzed by mass spectrometry, the method comprising: (a)dissolving the analyte in a suitable solvent; (b) injecting the saidanalyte solution into a atmospheric-pressure ionization source device asdescribed in claim 1; (c) causing the analyte solution to be vaporizedand heated; (d) causing the vaporized and heated analyte solution toimpact onto an active surface; (e) causing the ionized analyte to becollected by the analyzer or filter of a mass spectrometer.
 23. Themethod according to claim 22, wherein the analyte is dissolved in adipolar solvent.
 24. The method according to claim 23, wherein thesolvent is selected from H₂O, an alcohol optionally methanol or ethanol,or acetonitrile.
 25. The method according to claim 22, wherein the saidactive surface is inclined of an angle defining an impact angle for thesaid vaporized and heated analyte solution, wherein the said impactangle onto the active surface is 45° or less.
 26. The method accordingto claim 22, wherein the analyte solution is heated at a temperature inthe range of from 200° C. and 450° C., optionally from 250° C. and 350°C.
 27. The method according to claim 22, wherein a potential differenceof between 0 and 1000 V, in absolute value, is applied to the saidactive surface.
 28. The method according to claim 27, wherein the saidpotential difference, in absolute value, is of between 0 and 500 V,optionally between 0 and 200 V.
 29. The method according to claim 22,wherein the said analyte solution contains further an amino acid,optionally selected from the group consisting of glycine, lysine,istidine, aspartic acid and glutammic acid.
 30. The atmospheric-pressureionization source device of claim 1, wherein said inlet assembly isoperative to supply neutral molecules in gas phase to saidatmospheric-pressure ionization chamber.